Transforming growth factor-β enhances Rho-kinase activity and contraction in airway smooth muscle via the nucleotide exchange factor ARHGEF1

Yasin Shaifta; Charles E. MacKay; Nneka Ifeoma Irechukwu; Katie O'Brien; David B. Wright; Jeremy P. T. Ward; Gregory A. Knock

doi:10.1113/JP275033

Transforming growth factor-β enhances Rho-kinase activity and contraction in airway smooth muscle via the nucleotide exchange factor ARHGEF1

Yasin Shaifta, Charles E. MacKay, Nneka Ifeoma Irechukwu, Katie O'Brien, David B. Wright, Jeremy P. T. Ward, Gregory A. Knock

King's College London

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Abstract

Transforming growth factor-β (TGF-β), RhoA/Rho-kinase and Src-family kinases (SrcFK) have independently been implicated in airway hyper-responsiveness, but how they interact in order to regulate airway smooth muscle contractility is not fully understood. We found that TGF-β pre-treatment enhanced acute contractile responses to bradykinin (BK) in isolated rat bronchioles, and inhibitors of RhoGEFs (Y16) and Rho-kinase (Y27632), but not the SrcFK inhibitor PP2 prevented this enhancement. In cultured human airway smooth muscle cells (hASMC), TGF-β pre-treatment enhanced the protein expression of the Rho guanine nucleotide exchange factor ARHGEF1, MLC20, MYPT-1 and the actin-severing protein cofilin, but not of RhoA, ROCK2 or c-Src. In hASMC, acute treatment with BK triggered sub-cellular translocation of ARHGEF1 and RhoA and enhanced auto-phosphorylation of SrcFK and phosphorylation of MYPT1 and MLC20, but induced de-phosphorylation of cofilin. TGF-β pre-treatment amplified the effects of BK on RhoA translocation and MYPT1/MLC20 phosphorylation, but suppressed the effects of BK on RhoA-GTP content, SrcFK auto-phosphorylation and cofilin de-phosphorylation. In hASMC, an ARHGEF1 siRNA suppressed the effects of BK and TGF-β on RhoA-GTP content, RhoA translocation and MYPT1 and MLC20 phosphorylation, but minimally influenced the effects of TGF-β on cofilin expression and phosphorylation. ARHGEF1 expression was also enhanced in ASMC of asthmatic patients and in lungs of OVA-sensitized mice. Our data indicate that TGF-β enhances BK-induced contraction, RhoA translocation and Rho-kinase activity in airway smooth muscle largely via ARHGEF1, but independently of SrcFK and total RhoA-GTP content. A role for smooth muscle ARHGEF1 in asthmatic airway hyper-responsiveness is worthy of further investigation.

Original language	English
Pages (from-to)	47-66
Journal	The Journal of Physiology
Volume	595
Issue number	1
Early online date	25 Oct 2017
DOIs	https://doi.org/10.1113/JP275033
Publication status	Published - 1 Jan 2018

Keywords

Airway smooth muscle
TGF-beta
Rho-kinase
ARHGEF1

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Transforming growth factor-β_SHAIFTA_Accepted19October2017_GOLD VoRFinal published version, 2.55 MBLicence: CC BY

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title = "Transforming growth factor-β enhances Rho-kinase activity and contraction in airway smooth muscle via the nucleotide exchange factor ARHGEF1",

abstract = "Transforming growth factor-β (TGF-β), RhoA/Rho-kinase and Src-family kinases (SrcFK) have independently been implicated in airway hyper-responsiveness, but how they interact in order to regulate airway smooth muscle contractility is not fully understood. We found that TGF-β pre-treatment enhanced acute contractile responses to bradykinin (BK) in isolated rat bronchioles, and inhibitors of RhoGEFs (Y16) and Rho-kinase (Y27632), but not the SrcFK inhibitor PP2 prevented this enhancement. In cultured human airway smooth muscle cells (hASMC), TGF-β pre-treatment enhanced the protein expression of the Rho guanine nucleotide exchange factor ARHGEF1, MLC20, MYPT-1 and the actin-severing protein cofilin, but not of RhoA, ROCK2 or c-Src. In hASMC, acute treatment with BK triggered sub-cellular translocation of ARHGEF1 and RhoA and enhanced auto-phosphorylation of SrcFK and phosphorylation of MYPT1 and MLC20, but induced de-phosphorylation of cofilin. TGF-β pre-treatment amplified the effects of BK on RhoA translocation and MYPT1/MLC20 phosphorylation, but suppressed the effects of BK on RhoA-GTP content, SrcFK auto-phosphorylation and cofilin de-phosphorylation. In hASMC, an ARHGEF1 siRNA suppressed the effects of BK and TGF-β on RhoA-GTP content, RhoA translocation and MYPT1 and MLC20 phosphorylation, but minimally influenced the effects of TGF-β on cofilin expression and phosphorylation. ARHGEF1 expression was also enhanced in ASMC of asthmatic patients and in lungs of OVA-sensitized mice. Our data indicate that TGF-β enhances BK-induced contraction, RhoA translocation and Rho-kinase activity in airway smooth muscle largely via ARHGEF1, but independently of SrcFK and total RhoA-GTP content. A role for smooth muscle ARHGEF1 in asthmatic airway hyper-responsiveness is worthy of further investigation.",

keywords = "Airway smooth muscle, TGF-beta, Rho-kinase, ARHGEF1",

author = "Yasin Shaifta and MacKay, {Charles E.} and Irechukwu, {Nneka Ifeoma} and Katie O'Brien and Wright, {David B.} and Ward, {Jeremy P. T.} and Knock, {Gregory A.}",

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doi = "10.1113/JP275033",

language = "English",

volume = "595",

pages = "47--66",

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T1 - Transforming growth factor-β enhances Rho-kinase activity and contraction in airway smooth muscle via the nucleotide exchange factor ARHGEF1

AU - Shaifta, Yasin

AU - MacKay, Charles E.

AU - Irechukwu, Nneka Ifeoma

AU - O'Brien, Katie

AU - Wright, David B.

AU - Ward, Jeremy P. T.

AU - Knock, Gregory A.

PY - 2018/1/1

Y1 - 2018/1/1

N2 - Transforming growth factor-β (TGF-β), RhoA/Rho-kinase and Src-family kinases (SrcFK) have independently been implicated in airway hyper-responsiveness, but how they interact in order to regulate airway smooth muscle contractility is not fully understood. We found that TGF-β pre-treatment enhanced acute contractile responses to bradykinin (BK) in isolated rat bronchioles, and inhibitors of RhoGEFs (Y16) and Rho-kinase (Y27632), but not the SrcFK inhibitor PP2 prevented this enhancement. In cultured human airway smooth muscle cells (hASMC), TGF-β pre-treatment enhanced the protein expression of the Rho guanine nucleotide exchange factor ARHGEF1, MLC20, MYPT-1 and the actin-severing protein cofilin, but not of RhoA, ROCK2 or c-Src. In hASMC, acute treatment with BK triggered sub-cellular translocation of ARHGEF1 and RhoA and enhanced auto-phosphorylation of SrcFK and phosphorylation of MYPT1 and MLC20, but induced de-phosphorylation of cofilin. TGF-β pre-treatment amplified the effects of BK on RhoA translocation and MYPT1/MLC20 phosphorylation, but suppressed the effects of BK on RhoA-GTP content, SrcFK auto-phosphorylation and cofilin de-phosphorylation. In hASMC, an ARHGEF1 siRNA suppressed the effects of BK and TGF-β on RhoA-GTP content, RhoA translocation and MYPT1 and MLC20 phosphorylation, but minimally influenced the effects of TGF-β on cofilin expression and phosphorylation. ARHGEF1 expression was also enhanced in ASMC of asthmatic patients and in lungs of OVA-sensitized mice. Our data indicate that TGF-β enhances BK-induced contraction, RhoA translocation and Rho-kinase activity in airway smooth muscle largely via ARHGEF1, but independently of SrcFK and total RhoA-GTP content. A role for smooth muscle ARHGEF1 in asthmatic airway hyper-responsiveness is worthy of further investigation.

AB - Transforming growth factor-β (TGF-β), RhoA/Rho-kinase and Src-family kinases (SrcFK) have independently been implicated in airway hyper-responsiveness, but how they interact in order to regulate airway smooth muscle contractility is not fully understood. We found that TGF-β pre-treatment enhanced acute contractile responses to bradykinin (BK) in isolated rat bronchioles, and inhibitors of RhoGEFs (Y16) and Rho-kinase (Y27632), but not the SrcFK inhibitor PP2 prevented this enhancement. In cultured human airway smooth muscle cells (hASMC), TGF-β pre-treatment enhanced the protein expression of the Rho guanine nucleotide exchange factor ARHGEF1, MLC20, MYPT-1 and the actin-severing protein cofilin, but not of RhoA, ROCK2 or c-Src. In hASMC, acute treatment with BK triggered sub-cellular translocation of ARHGEF1 and RhoA and enhanced auto-phosphorylation of SrcFK and phosphorylation of MYPT1 and MLC20, but induced de-phosphorylation of cofilin. TGF-β pre-treatment amplified the effects of BK on RhoA translocation and MYPT1/MLC20 phosphorylation, but suppressed the effects of BK on RhoA-GTP content, SrcFK auto-phosphorylation and cofilin de-phosphorylation. In hASMC, an ARHGEF1 siRNA suppressed the effects of BK and TGF-β on RhoA-GTP content, RhoA translocation and MYPT1 and MLC20 phosphorylation, but minimally influenced the effects of TGF-β on cofilin expression and phosphorylation. ARHGEF1 expression was also enhanced in ASMC of asthmatic patients and in lungs of OVA-sensitized mice. Our data indicate that TGF-β enhances BK-induced contraction, RhoA translocation and Rho-kinase activity in airway smooth muscle largely via ARHGEF1, but independently of SrcFK and total RhoA-GTP content. A role for smooth muscle ARHGEF1 in asthmatic airway hyper-responsiveness is worthy of further investigation.

KW - Airway smooth muscle

KW - TGF-beta

KW - Rho-kinase

KW - ARHGEF1

U2 - 10.1113/JP275033

DO - 10.1113/JP275033

M3 - Article

SN - 0022-3751

VL - 595

SP - 47

EP - 66

JO - The Journal of Physiology

JF - The Journal of Physiology

IS - 1

ER -

Transforming growth factor-β enhances Rho-kinase activity and contraction in airway smooth muscle via the nucleotide exchange factor ARHGEF1

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Keywords

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